1. Field of the Invention
The invention concerns a process for the purification of synthetic oligonucletides which shortens strikingly the purification of synthetic oligonucleotides which is at present very complicated.
2. Brief Description of the Prior Art
By means of the known solid phase methods it is possible to produce a large number of different oligonucleotides relatively quickly.
In the usual processes of the chemical oligonucleotide sythesis (for example on a polymeric carrier, of FIG. 1) 5'-tritylated nucleotide elements are linked step by step in a desired sequence with each other. At the end of such a synthesis different short fragments (interrupted sequences) are obtained apart from the desired completely protected oligonucleotide; these fragments carry either free hydroxyl groups or acetyl groups (truncated by a blocking reaction between the steps of chain extension) or trityl groups at the 5'-terminal in a very limited quantity. Depending on the applied synthesis method (phosphotriester, phosphite triester) different modified oligonucleotides and fragments are also obtained as by-products. However, these products must be subjected to a series of deprotection and purification steps. The time required for this purification is far in excess of the synthesis time.
The following scheme represents the sequence of purification steps which are now-a-days carried out in most of the DNA synthesis laboratories (cf H. G. Gassen and Anne Lang, "Chemical and Enzymatic Synthesis of Gene Fragments, a Laboratory Manual", Verlag Chemie, Weinheim, 1982):
1. partial deprotection and separation from the carrier PA0 2. concentration of the solution PA0 3. chromatographic purification of the tritylated oligonucleotide PA0 4. concentration of the solution PA0 5. acid-catalysed detritylation PA0 6. concentration of the solution PA0 7. high performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) PA0 8. desalification (desalting) or elution and desalification (desalting) PA0 9. concentration of the solution PA0 that the oligonucleotides (cleaved off from the carrier and still with the lipophilic protecting group) are adsorbed without concentrating the cleaving solution PA0 on a carrier with reversed phase properties and optionally ion exchange properties, PA0 that the oligonucleotides without any lipophilic protecting group are removed, PA0 that the protecting group is cleaved off from the adsorbed oligonucleotides, PA0 that the adsorbed oligonucleotides are washed again, PA0 that the adsorbed oligonucleotides are eluted from the carrier and PA0 that the eluted oligonucleotides are then further purified in a manner known per se.
Of these purification steps steps 2 and 4, i.e. the concentration of the respective solutions, are especially time consuming.
During the deprotection step 1 all protection groups are cleaved off except the lipophilic trityl group. Only the trityl group is to be found at the 5'-terminal of the desired oligonucleotide.
The hydrophobic properties of this trityl group allow a separation from the previously mentioned by-products and cleaved protection groups by means of an interaction with reversed phase material.